A composition for use in the treatment of intervertebral disc-related pain

ABSTRACT

The present invention relates to intervertebral disc-related pain, such as low back pain, chronic low back pain, neck-pain, chronic neck pain and coccygodynia. A composition for use in the treatment of intervertebral disc-related pain is provided. The composition comprises lactic acid and has a pH below 4. The composition is administered into a disc space comprising the nucleus pulposus of an intervertebral disc.

TECHNICAL FIELD OF INVENTION

The present invention relates to intervertebral disc-related pain, suchas low back pain, chronic low back pain, neck pain, chronic neck painand coccygodynia, and a composition for use in the treatment ofintervertebral disc-related pain.

BACKGROUND

Low back pain, e.g. chronic low back pain, is a common condition thataffects about 80% of the adult population during their lifetime. Lowback pain is not a specific disease with known pathophysiology, butrather a symptom with many causes. A direct cause, such as a tumor, afracture, or an infection, has been estimated to be known only inapproximately 5-10% of the patients. In the remaining 90-95% of thecases, low back pain is idiopathic, i.e. without known origin.

The structure in the back that seems mainly responsible for low backpain production is the intervertebral disc. An intervertebral disc isarranged between two adjacent vertebrae. The intervertebral disc istypically flexible and allows for motion between the adjacent vertebrae.It is formed by a ring of connective tissue that mainly comprisescollagen, and a semi-liquid center comprising e.g. collagen andproteoglycans. The ring is called annulus fibrosus and the center iscalled nucleus pulposus.

Already at the age of 20-30 years, the intervertebral disc of a humanstarts to undergo ageing, a process often called disc degeneration.During the ageing process the intervertebral disc may leak or herniateand produce symptoms like low back pain and sciatica. The ageing of theintervertebral disc usually ends at the age of 60-80 years. At thisstage, the intervertebral disc has been transformed to solid and denseconnective tissue. When this occurs, the intervertebral disc willtypically not produce symptoms anymore since it is less likely to leakor herniate. The ageing of the intervertebral disc further implies areduction in disc height and a reduction of mobility of the spine.

It is known that disc degeneration will induce annular tears that mayallow for communication between the center of the intervertebral discand the outer surface of the annulus fibrosus. Thus, substances, such asinflammatory agents, from the center of the intervertebral disc may leakout onto the outer surface of the annulus fibrosus. Receptors, which areusually silent and arranged on the outer surface of the annulusfibrosus, may then be activated by inflammatory agents typically presentin the center of the intervertebral disc during disc degeneration. Thismechanism is suggested as one mechanism responsible for low back pain.

Another mechanism that has been suggested to be responsible for low backpain is that there may be newly formed blood vessels and nerves thatgrow from the outer surface of the annulus fibrosus into the center ofthe intervertebral disc through the annular tears. It is assumed thatthese nerves may produce pain when the intervertebral disc moves andexerts pressure on the nerves. One method to inhibit and disrupt such aprogress is disclosed in US2007253930.

One common procedure for treating low back pain is by surgicalstabilization of a vertebral segment comprising an intervertebral disc,which intervertebral disc presumably is producing pain. The rationale isto reduce movements of the pain-producing intervertebral disc in orderto avoid the ingrowing nerves to be compressed and produce pain. Thissurgical treatment is, however, invasive, and not entirely satisfactory.

Another proposed procedure for treating low back pain, or rathersciatica, is by so-called chemonucleolysis, wherein an enzyme isinjected into an intervertebral disc in order to dissolve the nucleuspulposus thereby reducing the pressure exerted by the nucleus pulposusof the intervertebral disc on e.g. a nerve.

Further, another proposed procedure for treating low back pain is byrejuvenation, or regeneration, of the intervertebral disc byintroduction of e.g. cultivated disc cells and stem cells. However, itseems unlikely that the nutritionally deprived environment in the centerof the intervertebral disc would successfully ensure survival of newlyintroduced cells.

For instance, regeneration promoted by a fibrosing agent has beendisclosed in WO 2005/046746. WO 2005/046746 relates e.g. to a methodcomprising introducing into an intervertebral disc space of a patient inneed thereof, a therapeutically effective amount of a fibrosing agent ora composition comprising a fibrosing agent. The fibrosing agent inducesa fibrotic response at the intervertebral disc space of the patient,thereby providing the patient with a beneficial result. WO 2005/046746also relates to an injectable composition comprising a fibrosing agentand a bulking agent.

However, there is still a need in the art to provide a safe andsatisfactory procedure to more successfully treat low back pain.

SUMMARY OF THE INVENTION

The object of the present invention is to provide a composition for usein the treatment of intervertebral disc-related pain, such as low backpain, chronic low back pain, neck pain, chronic neck pain andcoccygodynia.

The composition for use in the treatment of intervertebral disc-relatedpain may be formulated such that it may be administered in atherapeutically effective amount by a local injection to anintervertebral disc.

The concept of the present invention is to reduce the intervertebraldisc-related pain by accelerating the ageing of an intervertebral discthereby rendering the intervertebral disc stiffer, e.g. bytransformation of the intervertebral disc into solid and denseconnective tissue. The transformation of an intervertebral disc intosolid and dense connective tissue makes it more stable, andconsequently, the intervertebral disc obtains a reduced range of motion.An intervertebral disc transformed into solid and dense connectivetissue will neither allow any fluid component to leak out from the discspace, e.g. onto the outer surface of the annulus fibrosus, nor allownerves to grow into the intervertebral disc.

Already in 1959, Carl Hirsch stated that: “Sooner or later a substancemay be found by which a degenerated disc could be transformed to denseconnective tissue.” in the article “Studies on the Pathology of Low BackPain” published in The Journal of Bone and Joint Surgery, Vol. 41B, No.2, p. 237-243, May 1959. Nevertheless, it seems like nobody has untilnow presented any such substance.

The inventor of the present invention has surprisingly found that thesubstance successfully could be lactic acid having a pH below 4. Thisfinding is particularly surprising in view of the prior art ratherfocusing on decreasing the amount of lactic acid, or lactate, inside anintervertebral disc causing pain. For instance, US 2012/0022425 A1discloses a method for reducing lactic acid within an intervertebraldisc by injecting a lactic acid inhibitor into the vertebral disc toinhibit production of lactic acid, and thereby alleviating back painfrom lactic acid burn. Further, WO 2013/092753 A1 reveals a compound ofindole derivatives for inhibiting lactate production in the treatment offor example chronic back pain.

Lactic acid is a carboxylic acid with the chemical formulaCH₃CH(OH)COOH. As shown in the formula (I) below, lactic acid may in anaqueous solution lose a proton from its carboxyl group, producing thelactate ion CH₃CH(OH)COO⁻. The mole fraction of lactic acid to lactateion is 1:1.

CH₃CH(OH)COOH (aq)↔CH₃CH(OH)COO⁻+H⁺  (I)

Lactic acid and lactate are naturally present in the human body.

The concentration of lactate ion in tissue water of a lumbarintervertebral disc of a patient with back pain has been measured to bewithin the range of from 1 mmol/L to nearly 12 mmol/L, typically in therange of from 2 mmol/L to 6 mmol/L. These measured values have beenpresented at page 5 and in FIG. 6 of the scientific article “Oxygen andlactate concentrations measured in vivo in the intervertebral discs ofpatients with scoliosis and back pain” by Bartels et al., published inSpine 23(1): pp. 1-8, 1998.

As seen in Table 1, the molecular weight of lactate ion is 89.07 g/mol.A molar concentration of 1 mmol lactate ion per litre tissue water inthe lumbar intervertebral disc thus corresponds to a mass concentrationof 89.07 mg/L. Similarly, a molar concentration of 12 mmol lactate ionper litre tissue water in the disc corresponds to a mass concentrationof 1067 mg/L.

In a human, the disc space of a lumbar intervertebral disc has a volumeestimated to be approximately from 1.5 mL to 3.0 mL.

In view of the above, the person skilled in the art could easilycalculate the amount of lactate, expressed in moles or grams, in thedisc. An example is given in Table 1.

TABLE 1 Approximate amounts of lactate ion in a lumbar intervertebraldisc of a patient with back pain. Observed lactate ion concentration inthe 1-12 mmol/L tissue water of a lumbar disc (L3-L4) of a patient withback pain Average volume of the disc space of a 1.5-3 mL lumbar disccomprising the tissue water Calculated moles of lactate ion in the0.0015-0.036 mmol tissue water Molar weight of lactate ion 89.07 g/molCalculated mass of lactate ion in the 0.134-3.21 mg tissue water

The lactic acid may interfere negatively with the function of the cellsof the intervertebral disc, in particular the cells that produce theproteoglycans necessary for preventing the disc from ageing.

Ageing of an intervertebral disc is initiated by a reduced supply ofnutrients and oxygen via diffusion from the blood vessels in theadjacent vertebrae and from surrounding structures. This will graduallyinduce an accumulation of metabolic waste products in the intervertebraldisc, such as in the nucleus pulposus. One kind of metabolic wasteproduct that may be present is lactic acid.

Lactic acid may contribute to several mechanisms that will rendercellular death in the intervertebral discs, such as intracellular fataccumulation, mitochondrial swelling, chromatin clumping, and liberationof excitotoxic glutamate.

Lactic acid may liberate PGE₂ causing inflammation and production ofconnective tissue. Further, lactic acid may stimulate liberation ofTGF-beta, which in turn stimulates fibroblasts to produce collagen.

Lactic acid may also contribute to disseminated intravascularcoagulation and consumption coagulopathy, which increases the tendencyof red blood cells to aggregate, forming “blood sludge” and makes redblood cells more rigid, in turn, increasing the viscosity of the bloodand impairing circulation in the small vessels.

Thus, an increase in the concentration of lactic acid in anintervertebral disc by administration of a composition comprising lacticacid into the disc space of the intervertebral disc would thereforeaccelerate the ageing of the disc and induce transformation of thenucleus pulposus into connective tissue.

Ageing of the intervertebral disc, including transformation of thenucleus pulposus into connective tissue, renders the intervertebral discstiffer, and by administering a composition comprising lactic acid theageing may be accelerated in a controllable way. Typically, theconcentration of lactic acid may be increased in an intervertebral disc,more specifically in the disc space, in order to accelerate the ageing.

The inventor has found that a composition comprising lactic acid andhaving a pH below 4 induces a marked transformation of theintervertebral disc, thus making it stiffer. The marked transformationhas been interpreted as an accelerated ageing of the intervertebral discby transformation of the nucleus pulposus to connective tissue.Consequently, the inventor expects improvements for a patient withregard to intervertebral disc-related pain if a composition comprisinglactic acid and having a pH below 4, is administered into the nucleuspulposus of the intervertebral disc resulting in an increasedconcentration of lactic acid inside the disc space.

The inventor does expect improvements for a patient with regard tointervertebral disc-related pain, such as neck pain, low back pain orcoccygodynia, upon administration of lactic acid having a pH below 4, ora composition comprising lactic acid and having a pH below 4 into a discspace of an intervertebral disc being, at least partly, responsible forthe intervertebral disc-related pain.

According to a first aspect of the invention, a composition for use inthe treatment of intervertebral disc-related pain is provided. Thecomposition comprises lactic acid and has a pH below 4. The compositionis administered into a disc space comprising the nucleus pulposus of anintervertebral disc.

Advantages of the composition for use in the treatment of intervertebraldisc-related pain according to the present invention is a safer and moreefficient treatment of intervertebral disc-related pain, further alsobeing less expensive and less invasive than the treatments, e.g.surgical treatment, known in the state of the art. Further, lactic acidis biocompatible. The body of a vertebrate, such as a human, is capableof handling, such as degrading, lactic acid, as these compounds arenatural compounds, such as waste products, present in the body of thevertebrate.

The inventor suggests that the nucleus pulposus in the disc space of anintervertebral disc may transform to solid and dense connective tissue,similar to the connective tissue of the annulus fibrosus, when acomposition for use in the treatment of intervertebral disc-related painaccording to the present invention is administered into the nucleuspulposus. For instance, also, blood clotting may take place during thetransformation of the nucleus pulposus into connective tissue, renderingthe intervertebral disc even more solid and dense. The increasedstiffness is expected to result in decreased pain.

According to an embodiment, the composition for use is administered inan amount effective to increase the concentration of lactic acid in thedisc space to above 12 mmol/L.

The composition for use may be administered in an amount effective toincrease the concentration of lactic acid in the disc space to aconcentration higher than the concentration occurring during naturalageing.

According to an embodiment, the composition for use has a concentrationof lactic acid of at least 12 mmol/L, for example within the range offrom 12 to 12000 mmol/L, such as from 100 to 10000 mmol/L, such as from500 to 5000 mmol/L, such as from 800 to 2000 mmol/L.

According to an embodiment, the composition for use has a pH below 3.5,such as below 3.0, such as below 2.5, such as below 2, and such as below1.5.

According to an embodiment, the composition for use is administered tothe disc space of an intervertebral disc contributing to theintervertebral disc-related pain.

In an example, the composition for use may be administered to any or allof the intervertebral discs which are suspected to contribute to theintervertebral disc-related pain.

According to an embodiment, lactic acid having a pH below 4 isadministered by local injection into the disc space comprising thenucleus pulposus.

The local injection may typically be performed by a syringe.

According to an embodiment, the lactic acid is administered in a singledosage within the range of from 2 mg to 200 mg, such as from 5 mg to 200mg, such as from 10 to 100 mg, such as from 10 to 50 mg, such as from 15to 30 mg. The single dosage corresponds to the amount of lactic acidbeing administered per disc space.

According to an embodiment, the composition for use comprising lacticacid and having a pH below 4 is administered at a single occasion in thesingle dosage.

According to an embodiment, the composition is in the form of an aqueoussolution comprising said lactic.

Typically, the composition for use in the treatment of intervertebraldisc-related pain is provided in a liquid state suitable for localinjection.

According to an embodiment, the intervertebral disc-related pain isselected from neck pain, chronic neck pain, low back pain, chronic lowback pain, and coccygodynia.

In some examples, the composition may further comprise at least oneagent selected from solubilizers, stabilizers, buffers, tonicitymodifiers, bulking agents, viscosity enhancers, viscosity reducers,surfactants, cheating agents, preservatives and adjuvants.

In a human, the amount of the composition to be administered may bewithin the range of from 0.05 mL to 5 mL, such as from 0.1 to 3 mL, e.g.from 0.2 mL to 2 mL. These amounts correspond more or less to the volumeof the nucleus pulposus in a human. For a lumbar intervertebral disc,the amount of the composition to be administered may be approximatelyfrom 1.5 mL to 3.0 mL. For a cervical intervertebral disc, the amount ofthe composition to be administered may be approximately 0.5 mL. For acoccygeal intervertebral disc, the amount of the composition to beadministered may be approximately 0.2 mL.

By the term “single occasion” is herein meant at a single visit at amedical office, such as during a visit to the doctor e.g. at a hospital.The visit may be no longer than 24 hours, such as from 0.5 to 5 hours.The term typically, but not necessarily, implies that the single dosageis administered by only a single injection at the single occasion.However, the term also covers cases where the single dosage isadministered at a single occasion but by several injections, such asfrom 2 to 10 injections per single occasion, e.g. from 2 to 5 injectionsper single occasion.

By the term “repeated occasions” is herein meant at more than one visit,i.e. a plurality of visits, at a medical office, such as during morethan one visit to the doctor e.g. at a hospital. Each visit may be nolonger than 24 hours, such as from 0.5 to 5 hours. The term typically,but not necessarily, implies that the single dosage is administered byonly a single injection but at repeated occasions. However, the termalso covers cases where the single dosage is administered at repeatedoccasions but by several injections, such as from 2 to 10 injections pereach of said repeated occasions, e.g. from 2 to 5 injections per each ofsaid repeated occasions.

By the term “intervertebral disc” is meant an element lying between twoadjacent vertebrae in the spine. Each intervertebral disc forms acartilaginous joint to allow slight movement of the vertebrae, and actsas a ligament to hold the vertebrae together. An intervertebral discconsists of an outer annulus fibrosus, which surrounds an inner nucleuspulposus. A human vertebral column comprises 23 intervertebral discs: 6in the neck (cervical region), 12 in the middle back (thoracic region),and 5 in the lower back (lumbar region). In addition, intervertebraldiscs are also arranged between the coccygeal bones. An intervertebraldisc may also be called a disc.

By the term “nucleus pulposus” is meant the jelly-like substance in themiddle of an intervertebral disc. The nucleus pulposus compriseschondrocyte-like cells, collagen fibrils, and proteoglycan aggrecansthat aggregate through hyaluronic chains. Attached to each aggrecanmolecule are the glycosaminoglycan (GAG) chains of chondroitin sulfateand keratan sulfate. The nucleus pulposus acts as a shock absorber, andkeeps the two adjacent vertebrae separated.

By the term “annulus fibrosus” is meant, a lamina of fibrous tissue andfibrocartilage formed as at the circumference of the nucleus pulposus.The annular fibrosus serves to distribute pressure evenly across theintervertebral disc.

By the term “disc space” is meant the space of an intervertebral discwhich is filled by the nucleus pulposus and which has a circumferencedefined by the annular fibrosus.

By the term “cranial endplate” is meant the surface of an intervertebraldisc facing towards the cranium. The cranial endplate is arranged onopposite side of the intervertebral disc compared to the caudalendplate.

By the term “caudal endplate” is meant the surface of an intervertebraldisc facing away from the cranium. The caudal endplate is arranged onopposite side of the intervertebral disc compared to the cranialendplate.

By the term “facet joint” is meant a paired articular structuretypically having a joint surface which is covered with articularcartilage. The facet joint is typically enclosed by a capsule. The facetjoint form an articulation between the inferior articular process of thevertebrae and the superior articular process of the vertebrae. A facetjoint is typically constructed to allow movement and to providemechanical support to the vertebral column.

By the term “transverese process” is meant a bony formation that extendslaterally from the vertebral arch on both sides. It is also termedprocessus costarius.

By the term “intervertebral disc-related pain” is herein meant a painrelated to a pain-producing intervertebral disc. Intervertebraldisc-related pain may be pain related to at least one of a cervicalvertebra (C), a lumbar vertebra (L), a sacral vertebra (S) and acoccygeal vertebra (Co). Examples of intervertebral disc-related painmay be low back pain, chronic low back pain, neck pain, chronic neckpain and coccygodynia.

By the term “chronic low back pain” is meant low back pain whereinsymptoms have occurred during more than 12 weeks.

By the term “chronic neck pain” is meant neck pain wherein symptoms haveoccurred during more than 12 weeks.

By the term “coccygodynia” is meant pain in the coccyx or tailbone area.

By the term “flexion stiffness” is herein meant a characteristicdescribing the stiffness of an intervertebral disc arranged in a segmentof a vertebral column. The flexion stiffness may be determined byapplying a force to the segment of a vertebral column until it reaches afull lateral flexion mode, and by, thereafter, measuring the distancebetween the transverse processes of the vertebras being arranged on thetwo opposite sides of the intervertebral disc, respectively. The fulllateral flexion mode is defined as the state where the intervertebraldisc of the segment of the vertebral column cannot be forced furtherwithout breaking of the segment of the vertebral column. Thischaracteristic is measured in millimeter. The flexion stiffness is a wayof characterizing the flexural rigidity of the segment of the vertebralcolumn, and more specifically, the flexural rigidity of theintervertebral disc.

Flexural rigidity is generally defined as the force couple required tobend a non-rigid structure to a unit curvature. It is a measure ofstiffness of a structural member; the product of modulus of elasticityand moment of inertia divided by the length of the member. In otherwords, it is the ratio of stress to strain in an elastic material whenthat material is being bent.

According to a second aspect, there is provided a method for treatmentof intervertebral disc-related pain by administration of atherapeutically effective amount of lactic acid having a pH below 4 intothe nucleus pulposus of an intervertebral disc of a patient in needthereof. Effects and features of this second aspect of the presentinvention are analogous to those described above in relation to thefirst aspect of the present invention.

According to a third aspect, there is provided use of lactic acid havinga pH below 4 in the manufacture of a medicament for the treatment ofintervertebral disc-related pain. Effects and features of this thirdaspect of the present invention are analogous to those described abovein relation to the previous aspects of the present invention.

According to a fourth aspect, there is provided lactic acid having a pHbelow 4 for use in the treatment of intervertebral disc-related pain.Effects and features of this fourth aspect of the present invention areanalogous to those described above in relation to the previous aspectsof the present invention.

Further features of, and advantages with, the present invention willbecome apparent when studying the appended claims and the followingdescription. The skilled person realizes that different features of thepresent invention may be combined to create embodiments other than thosedescribed in the following, without departing from the scope of thepresent invention.

BRIEF DESCRIPTION OF THE DRAWINGS

These and other aspects of the present invention will now be describedin more detail, with reference to the appended drawings showingembodiment(s) of the invention.

In FIG. 1, a cross section of a vertebral column of a human isschematically shown.

In FIG. 2, two adjacent vertebras of a human vertebral column areschematically shown in a side view.

In FIG. 3, a lower part of a vertebral column of a human isschematically shown in a side view.

In FIG. 4, a vertebral segment is schematically shown in a posteriorview.

In FIG. 5, it is schematically shown how the anterio-posterior length ofa cross-section of an intervertebral disc is measured.

In FIG. 6, it is schematically shown how the bilateral width of across-section of an intervertebral disc is measured.

FIG. 7 shows three intervertebral discs in cross-section, and thetransformation of the nucleus pulposus into connective tissue in thedisc administered with a composition according to an embodiment of thepresent invention.

FIG. 8 shows the experimental results from the study of collagenproduction in fibroblasts upon treatment with lactic acid.

FIG. 9 shows the experimental results from the study of collagenproduction in nucleus pulposus cells upon treatment with lactic acid.

FIG. 10 shows the experimental results from the study of collagenproduction by human fibroblasts upon treatment with lactic acid withdifferent pH values.

FIGS. 11-15 show the experimental results of example 2 below.

As illustrated in the figures, the sizes of layers and regions areexaggerated for illustrative purposes and, thus, are provided toillustrate the general structures of embodiments of the presentinvention. Like reference numerals refer to like elements throughout.

DETAILED DESCRIPTION OF THE INVENTION

As a vertebrate ages, its intervertebral discs undergo a transformation.An effect of the transformation is that the nucleus pulposus begins todehydrate and the concentration of proteoglycans in the matrixdecreases, resulting is a decreased size of the intervertebral disc.Another effect is that the annulus fibrosus becomes weaker and has anincreased risk of tearing. The effects of the transformation of theintervertebral disc may cause intervertebral disc-related pain, e.g.neck pain, low back pain or coccygodynia, in the state before theintervertebral disc gets sufficiently solid and dense.

A vertebral column of a vertebrate comprises vertebrae, which surroundand protect a spinal cord. In humans, the vertebral column is situatedin the dorsal aspect of torso. Between two adjacent vertebrae, anintermediate intervertebral disc is arranged, i.e. the vertebrae arealternated by intervertebral discs forming the vertebral column. Thespecific structure and further parts of the vertebral column are knownto a person skilled in the art.

FIG. 1 schematically shows a cross section of a vertebral column 100 ofa human. Adjacent to a vertebral body 15 of a vertebra, anintervertebral disc comprising an annulus fibrosus 10 and a nucleuspulposus 11 is arranged. The nucleus pulposus 11 fills up the so-calleddisc space of the intervertebral disc. The annulus fibrosus 10 surroundsthe nucleus pulposus 11 and defines the border of the nucleus pulposusas well as of the disc space.

A spinal cord 17 is situated in the centre of the vertebral column, andadjacent to the intervertebral disc. Spinal nerves 16, 16′, extend outfrom the spinal cord 17 to opposite sides of and closely to theintervertebral disc.

A facet joint 14, 14′, is situated between an inferior articular process13, 13′ and a superior articular process 12, 12′. On opposite sides ofthe spinal cord 17, two facet joints 14, 14′, are arranged,respectively. The facet joints 14, 14′, are arranged in approximatelythe same cross-section and plane.

FIG. 2 schematically shows a segment of a vertebral column 200comprising two adjacent vertebras 20, 22. A first vertebra 22 and asecond vertebra 20 are arranged on opposite sides of an intervertebraldisc 21. The first vertebra 22 is arranged relatively closer to thethorax, and the second vertebra 20 is arranged relatively closer to thesacrum. The caudal endplate 23 of the first vertebra 22 and the cranialendplate 25 of the second vertebra 20 are shown in FIG. 2. The cranialendplate 25 and the caudal endplate 23 are facing opposite sides of theintervertebral disc 21.

FIG. 2 also schematically shows how a facet joint 24 is arranged betweenthe inferior articular process of the first vertebra 22 and the superiorarticular process of the second vertebra 20. A transverese process 26extends laterally from the vertebral arch.

FIG. 3 schematically shows a lower part of a vertebral column 300. Thecoccygeal vertebrae 36 of the vertebral column is arranged at an endportion of the lower part of the vertebral column 300. The sacrum 39 ofthe vertebral column is arranged adjacent to the coccygeal vertebrae 36,closer to the thorax than the coccygeal vertebrae 36. A fifth lumbarvertebra, herein called L5, 30 is arranged adjacent to the sacrum 39,closer to the thorax than the sacrum 39. In a direction from the sacrum39 towards the thorax, several vertebras are arranged in a row startingwith L5, 30. Adjacent to the fifth lumbar vertebra 30, i.e. L5, thefollowing vertebras are arranged in order: a fourth lumbar vertebra 32,i.e. L4, a third lumbar vertebra, i.e. L3, a second lumbar vertebra,i.e. L2, and a first lumbar vertebra 38, i.e. L1; the first lumbarvertebra being arranged relatively closest to the thorax. In betweeneach two adjacent vertebras, an intermediate disc 31 is arranged.Intervertebral discs (not shown) are also imposing the coccygealvertebrae 36.

EXAMPLES

No reliable animal model for measuring pain are available, instead endpoint flexion (e-g—flexural stiffness) and visual decrease in nucleusspace have been used. Due to arguments above (nerve ingrowth andleakage) it is likely that a solid disc, which reduces flexion andreduced nucleus space, will also relate to a decrease in pain, asexperienced by patients.

Example 1

The procedure for inducing and assessing accelerated transformation ofthe nucleus pulposus into connective tissue in an intervertebral disc ina pig by administration of a composition for use in the treatment ofintervertebral disc-related pain comprising lactic acid and having a pHbelow 4 will herein after be more fully described.

In the present example, a composition for use in treatment ofintervertebral disc-related pain comprising lactic acid and having a pHbelow 4 is administered into the nucleus pulposus of an intervertebraldisc arranged between the third lumbar vertebra L3 and the fourth lumbarvertebra L4. A person skilled in the art could easily understand thatthe same procedure may be applied to any intervertebral disc in avertebral column.

Thus, the steps of the procedure are the following:

100. preparing a composition comprising lactic acid having a pH below 4;

101. anaesthetizing a pig comprising a vertebral column comprising theintervertebral disc comprising the nucleus pulposus into which thecomposition is to be administered;

102. allow access to the intervertebral disc through a lateral incisionbetween the lowest rib and the iliac crest of the pig;

103. incising the intervertebral disc;

104. administering, herein by locally injecting, the composition intothe nucleus pulposus by an injection needle;

105. allowing the pig to move freely for seven days after recoveringfrom anaesthesia;

106. harvesting the lumbar spine en bloc, the harvested segmentcomprising the vertebral bodies and the intervertebral disc comprisingthe nucleus pulposus subjected to injection, but without the posteriorelements (the vertebral arch and facet joints);

107. measuring the distance between the transverse processes at thelevels of discs L 2-3, L 3-4, L4-5 without any external force applied;

108. applying an external force to the segment of the vertebral columnuntil a full lateral flexion mode is achieved for the lumbar spinespecimen;

109. measuring the distance between the transverse processes at thelevels of discs L 2-3, L 3-4, L4-5 under full lateral flexion;

110. performing a cross-section of the discs and measuring the length(anterio-posterior direction) and the width (bilateral direction) of thedisc space.

Preparation of a Composition Comprising Lactic Acid

A pure solution of lactic acid was purchased from Sigma Aldrich (productnumber: 69775 Fluka; CAS number: 50-21-5, Stockholm, Sweden). As shownin Table 2, the molecular weight of lactic acid is 90.08 g/mol and thedensity of the pure solution from Sigma Aldrich was 1.209 g/mL,respectively.

The concentration of lactic acid in the pure solution from Sigma Aldrichwas consequently calculated to be 0.0134 mol/mL, equals 13.4 mol/L.

The pure solution of lactic acid was thereafter diluted 10 times usingdistilled water at room temperature. More explicitly, 1 mL of the puresolution of lactic acid from Sigma Aldrich was diluted with 9 mL ofdistilled water. The resulting concentration of lactic acid in theprepared composition was consequently 1.34 mol/L, and had a pH ofapproximately 1.8.

TABLE 2 Amounts of lactic acid in the prepared composition. Density oflactic acid in the pure lactic acid 1.209 g/mL solution Molar weight oflactic acid 90.08 g/mol Calculated concentration of lactic acid in 13.4mol/L the pure lactic acid solution Degree of dilution 10% Calculatedconcentration of lactic acid in 1.34 mol/L the diluted pure lactic acidsolution

Administration of a Composition Comprising Lactic Acid to the NucleusPulposus of an Intervertebral Disc in a Pig by Local Injection

Two pigs were anesthetized and placed on their right side. Access to theL4-5 intervertebral disc was obtained through a lateral incision betweenthe lowest rib and the iliac crest on the left side of each pig.Thereafter, the L3-4 intervertebral disc was incised with a scalpel.

The composition comprising lactic acid was injected by a syringe intothe nucleus pulposus of the L3-4 intervertebral disc. The compositioncomprising lactic acid in a total concentration of 1.34 mol/L with a pHof 1.8 was injected in an amount of approximately 0.2 mL into thenucleus pulposus, as shown in Table 3. The composition was injected in asingle step at a single occasion.

Both pigs seemed to tolerate the procedure well and no adverse reactionsuch as decreased mobility or vocalization was observed during theperiod of seven days until harvest. At harvest, the pigs were killed.

TABLE 3 Amount of lactic acid in the composition administered to a L3-4interverebral disc of a pig. Volume of injected diluted pure lactic acid0.2 mL solution Calculated moles of lactic acid in the 0.268 mmolinjection Calculated mass of lactic acid in the 23.9 mg injectionAssessment of Transformation of Nucleus Pulposus into Connective Tissuein an Intervertebral Disc Administered with a Composition ComprisingLactic Acid

The injection site was observed by the naked eye. No adverse reaction atthe injection site, such as bleedings, inflammation or necrosis, wasobserved in any of the pigs. The segment of the vertebral columnextending from the lumbar vertebra L2 to the sacral vertebra S1 wasremoved. The facet joints were removed, thus allowing full flexibilityof the discs with no restraints from other structures.

A—Flexion Stiffness of the Intervertebral Disc Before and afterAdministration of the Composition, Respectively

In FIG. 4, a segment of a vertebral column comprising intervertebraldiscs 21, namely the intervertebral discs L2-3, L3-4 and L4-5, is shown.

During the assessment of flexion stiffness, the distance between each ofthe respective adjacent transverse processes 26 of the vertebral column,thus, the segment of the vertebral column extending from the lumbarvertebra L2 to the sacral vertebra S1, was measured by calipers when thesegment of the vertebral column was arranged in a mode without anyexternal load applied.

Thereafter, the vertebral column, thus, the segment of the vertebralcolumn extending from the lumbar vertebra L2 to the sacral vertebra S1,was manually forced into a full lateral flexion mode by applying anexternal force to each of the two end portions of the part of thevertebral column until a critical limit was met, i.e. until the fullflexion mode was achieved. The motion of the transverse processes uponan applied force is schematically shown in FIG. 4 by arrows with dottedlines.

The critical limit was defined as the point just before the breakingpoint of the segment of the vertebral column. Thus, the external forcewas applied such that a maximum lateral flexion was obtained withoutbreaking any part of the vertebral segment.

The force was assumed to be similar for the segment of the vertebralcolumn in each of the both pigs. In the position of full lateralflexion, the distance between the adjacent transverse processes for thediscs L 2-3, L 3-4, L4-5 were measured by calipers.

The distance between the adjacent transverse processes for a certaindisc in the mode without external load was subtracted from the distancebetween the same transverse processes in the mode with an external loadapplied to achieve full lateral flexion mode, thereby providing a valueof the balanced distance obtained by the full lateral flexion. Thebalanced value for the injected intervertebral disc reflects the flexionstiffness of the intervertebral disc being treated with a compositionfor use in the treatment of intervertebral disc-related pain compared toa non-injected intervertebral disc.

The flexion stiffness is an indirect measure of the transformation ofthe nucleus pulposus into connective tissue, as the smaller the balancedvalue, the stiffer the intervertebral disc. The stiffer theintervertebral disc, the higher the content of solid and denseconnective tissue. Hence, the flexion stiffness indicates whether thenucleus pulposus has undergone a transformation into connective tissue,i.e. an accelerated ageing, or not.

The measurements show that the injected discs (L 3-4) had a much smallerbalanced value than the non-injected adjacent discs (L 2-3; L 4-5),which indicates a higher flexion stiffness of the injected disc. Thus,an accelerated transformation of the nucleus pulposus into connectivetissue had taken place inside the disc space of the injected disccompared to inside the disc spaces of the non-injected discs (see Table4).

TABLE 4 Difference in distance between transverse processes before andupon full lateral flexion (mm ± SD). Intervertebral disc Distance (mm) L2-3 3.1 ± 1.1 L 3-4 0.3 ± 0.6 L 4-5 2.7 ± 0.9

b—Dimension of the Disc Space Before and after Administration of theComposition, Respectively

The intervertebral discs (L2-3, L3-4, L4-5) were cross-sectioned and thelength of the disc space (anterio-posterior direction) and the width(bilateral direction) of the disc space were measured by calipers.

In FIGS. 5 and 6, an intervertebral disc is schematically shown incross-section. The intervertebral disc comprises the annulus fibrosus10, and a disc space defined by the annulus fibrosus and comprising thenucleus pulposus 11.

In FIG. 5, an arrow schematically shows how the anterio-posterior lengthof the disc space of the intervertebral disc is measured. In FIG. 6, anarrow schematically shows how the bilateral width of the disc space ofthe intervertebral disc is measured.

As seen from the measurements, the average anterio-posterior length ofthe disc space was significantly lower in the injected discs (L 3-4)than in the adjacent non-injected discs (L2-3, L4-5) (see Table 5).

TABLE 5 Average antero-posterior length of the disc space (mm ± SD).Intervertebral disc Length (mm) L 2-3 22.0 ± 1.0 L 3-4 13.5 ± 1.3 L 4-521.3 ± 2.2

As seen from the measurements, the average bilateral width of the discspace was significantly lower in the injected discs (L 3-4) than in theadjacent non-injected discs (L2-3, L4-5) (see Table 6).

TABLE 6 Average bilateral width of the disc space (mm ± SD).Intervertebral disc Width (mm) L 2-3 8.4 ± 0.7 L 3-4 4.0 ± 1.4 L 4-5 8.6± 2.3

FIG. 7 shows the intervertebral discs L2-3, L-3-4 and L4-5,respectively, from one of the pigs in the experiments above. Width anddepth of the disc space are indicated by bold straight linescorresponding to the schematic drawings in FIG. 5-6.

In FIG. 7, it is further shown how the disc space of the injected L3-4disc had a much smaller cross-sectional area than the non-injected L2-3and L4-5 discs, respectively. Thus, it can be verified by the naked eyethat there was newly formed connective tissue in the disc space formercomprising nucleus pulposus in the injected intervertebral discs.

Conclusion of Example 1

It is evident that the disc space in the two non-injected discs (L2-3)and (L4-5) is much deeper and wider than the disc (L3-4) which had beenadministered with the composition comprising lactic acid with a pH of1.8. It seems that the former disc space has been exchanged with newlyformed connective tissue (emphasized by the fading, additional lines inan arc-like arrangement, in FIG. 7), such that the annular fibrosus(formed by a ring of connective tissue that mainly comprises collagen)has expended at the expense of the nucleus pulposus that has decreasedin size.

Thus, a flexion stiffness of the injected intervertebral discs isachieved, and the stiffness can suppress the pain experienced by apatient having intervertebral disc-related pain. An advantage of thisway of treatment of intervertebral disc-related pain, is that thetreatment is less invasive than current treatment methods, such ascompared to the current treatment method of arthrodesis.

In the example described above, the intervertebral disc was arranged inthe lumbar spine. However, a similar process is expected to be observedin an intervertebral disc arranged in the cervical spine or in thecoccygeal spine.

Example 2

In the present example, a comparison has been made between the use of acomposition comprising lactic acid and having a pH below 4 (below namedactive injection), and a placebo injection having a pH below 4 foradministration into the nucleus pulposus of an intervertebral discarranged between the third lumbar vertebra L3 and the fourth lumbarvertebra L4.

Methods and Formulations:

Eight pigs were anesthetized and placed on their right side. Through alateral incision the L3-4 disc was exposed. The disc was injected with atotal volume of 0.2 ml with either active injection or placeboinjection.

-   -   Active injection: 120 mg/ml lactic acid (PURAC PF 90 Batch Nr.:        1406001940)+180 mgl/ml lohexol (Histodenz CAS#66108-95-0 LOT        #WXBB5310V) in a saline solution (0.9% NaCl), the pH was        measured to 1.5    -   Placebo injection: 180 mgl/ml lohexol in a saline solution (0.9%        NaCl), the pH was adjusted to 1.5 with HCl (Hydrochloric acid,        Titrisol).

After four weeks the pigs were killed and the lumbar spine washarvested. All musculature was removed as well as the facet joints andligamentum flavum between the L2-3, L3-4 and L4-5 vertebrae. This wasperformed to enable an assessment of flexion. The following endpointswere measured:

-   -   A. The distance between the transverse processes was measured by        callipers at full contralateral and ipsilateral flexion.    -   B. The disc space was measured anterior posterior and bilateral        with a calliper    -   C. X-ray pictures were taken at the day of injection and at the        harvest day to evaluate that the injection was correctly placed        and if the discs were radio opaque after four weeks.

Results:

A) Decrease in Lateral Flexion:

A significant difference in flexion can be observed between activetreated and placebo treated discs (FIG. 11). This clearly indicates thatlactic acid and not the iohexol, the pH or the injection per se, willinduce this decrease in flexion. The results from the study arestatistically significant.

B) Decrease in Nucleus Space:

In FIGS. 12a-c , it is obvious that the disc space is similar in sizebetween an untreated disc, FIG. 12a , and a placebo-injected disc, FIG.12b , whereas the treated disc, FIG. 12c , has a significantly smallersize. The absolute numbers are presented in FIGS. 13 and 14. The resultsfrom the study are statistically significant.

C) No Decrease in Visual Disc Height after Injection.

A known issue relating to the natural aging of the disc is decrease indisc height due to the degeneration of the disc. An assumption is thatdue to the rapid process of the transformation of the disc caused by thetreatment we estimate that the geometry of the disc will be fixed and noor very little disc height will be lost. This hypothesis was verifiedwith visual analysis of the x-ray pictures of the injected disc (L3-L4)4 weeks after the treatment. As shown in FIG. 15, no difference in discheight between treated and untreated discs can be seen.

Conclusions of Example 2:

The conclusions from the in vivo study in example 2 are that thetreatment effectively transforms the nucleus pulposus to connectivetissue causing a decrease in flexion and a drastic decrease in discspace without any observed reduction in disc height. The effect isstatistically significant compared to placebo. It may also concludedthat a pH adjustment per se does not have any effect.

The results from the study thus provide proof of concept that theproduct effectively transforms the disc space into connective tissue.

To observe the effects of lactic acid on a cell level, studies wereconducted on fibroblasts, commonly present in connective tissue such asthe annulus fibrosus, and nucleus pulposus cells, commonly present inthe nucleus pulposus, respectively. As a measure on how the cellstransformed in response to treatment of lactic acid, the collagenproduction in the cells was studied.

Example 3

Study of Collagen Production in Fibroblasts Upon Treatment with LacticAcid

Culture of Adult Human Dermal Fibroblasts (HDFa)

Human dermal fibroblasts isolated from adult skin, so-called HDFa, (LifeTechnologies Frederick, USA) were cultured and studied. Mature humanintervertebral disc cells have been described as being fibrocytic (orfibroblast-like) in the outer annulus fibrosus. Fibroblasts are the mostcommon type of cell found in connective tissue. Fibroblasts maynaturally secrete collagen proteins that are used to maintain astructural framework for many tissues and also play an important role inwound healing.

Firstly, cryopreserved fibroblasts were thawed in a 37° C. water bath.The thawed fibroblasts were then dispersed by using a 1 milliliterpipette to move the suspension of thawed fibroblasts up and down in thevial. The dispersed fibroblasts were then diluted in trypan bluesolution (Cat. No. 15250-061, Lot No. 1311086, Gibco Life Technologies),and the concentration of viable fibroblast was determined by ahemacytometer.

The dispersed fibroblasts were then diluted again, this time insupplemented Medium 106 to a concentration of 2.5×10⁴ viable fibroblastsper milliliter. 5 ml of fibroblast suspension was then added to a T25cell culture flask having a volume of 25 cm³ to achieve an initialdensity of 5.0×10³ viable fibroblasts per milliliter in the T25 flask byfurther dilution with supplemented Medium 106.

The supplemented Medium 106 consisted of Medium 106 (Cat. No. M-106-500,Life Technologies, Paisley, Great Britain) supplemented with Low SerumGrowth Supplement, LSGS, (Life Technologies, Paisley, Great Britain) ata concentration of fetal bovine serum of 2% by volume

The T25 flask comprising the prepared fibroblasts was swirled todistribute the fibroblasts in the medium. The cell culture wasthereafter incubated in a 37° C., 5% CO₂/95% air humidified cell cultureincubator for 72 hours.

At confluence, the fibroblasts were diluted in the supplemented media toavoid alternations in cell phenotype.

Preparation of Lactic Acid

Lactic acid (Fluka 69775, Sigma-Aldrich, Stockholm, Sweden) was weightedinto a sterile 10 mL tube or 50 mL tube. Milli-Q water (>18.2Ω) wasadded to prepare a stock solution of lactic acid. The stock solution wasmixed and stored before preparing final solutions of lactic acid withvarying concentrations. The period of storage was less than 1 hour atambient temperature, or, alternatively, less than 24 hours at atemperature of 4° C.

Effect of Lactic Acid on Collagen Production in Adult Human DermalFibroblasts (HDFa)

Fibroblasts cultured as described above were detached from the cellculture flask and placed on 6-well plates at an initial density of6.0×10⁴ viable cells per well. The fibroblasts were grown insupplemented Medium 106. The fibroblasts in some of the wells were alsotreated with lactic acid (Fluka 69775, Sigma-Aldrich, Stockholm, Sweden)in various concentrations: 0, 0.5, 2, 5, 10, 20 and 50 mg/mL,respectively. The fibroblasts were incubated in a 37° C., 5% CO₂/95% airhumidified cell culture incubator for 48 hours.

To study the effect of lactic acid on the collagen production in thefibroblasts, a spectrophotometric method called Soluble Collagen Assay(QuickZyme Biosciences, Leiden, Netherlands) based on binding of SiriusRed dye to collagen, was applied. The study was performed twice.

Cell media was collected from each well and 140 μL was pipetted into a96-well plate. Samples were taken in duplicates. Medium samples weremixed thoroughly with 60 μL Sirius Red dye solution by pipetting up anddown at least five times. The 96-well plate was centrifuged at 3000×gfor 1 hour. All of these steps were performed at a temperature below 25°C., for example the centrifugation was performed at 4° C.

The centrifuged sample was washed and the supernatant removed. The cellpellet was resuspended in 150 μL detection solution by thoroughly mixingby pipetting up and down at least ten times. Thereafter, 100 μL of eachsample was transferred into a new 96-well plate and collagen content wasmeasured spectrophotometrically at an optical density of 540 nm.

From the two studies each performed in duplicates, it was clearly shownthat addition of lactic acid to fibroblasts increases the averageproduction of collagen in the fibroblasts, as indicated in Table 7 andFIG. 8. The average production was measured after 2 days of treatmentwith lactic acid.

In FIG. 8, the results from the first set of the study are presented bydiamonds, while the results from the second set of the study arepresented by squares. A two-period moving average trendline has beenincluded to schematically show the trend in collagen production for eachset. The trendline for the first set is presented by a dotted line, andthe trendline for the second set is presented by a dashed line,respectively. The x-axis shows the concentration of the lactic acidadded to the wells comprising fibroblasts, and the y-axis shows theaverage amount of produced collagen in these wells upon measurementafter two days from addition of the lactic acid into the wells.

More particular, the increase in production of collagen was significantwhen lactic acid was added to a concentration of at least 2 mg/mL, suchas at least 5 mg/mL, in the well. Further, it was shown that thecollagen production increased with increasing lactic acid concentrationup to at least 20 mg/mL or at least 50 mg/mL as also indicated in FIG.8.

TABLE 7 Effect on collagen production in fibroblasts of lactic acid.2^(nd) set of 1^(st) set of study: study: Average Average amountStandard amount of Standard of produced deviation produced deviationConcentration collagen in the 1^(st) collagen in the 2^(nd) of lacticacid per well set of per well set of [mg/mL] pH [μg] study [μg] study 0— 0.033 0.1 0.0711 0.111 0.5 3.1 10.8 12 0.0995 0.0435 2 2.8 9.57 4 8.945.72 5 2.6 21.2 0.8 12.0 0.281 10 2.4 16.7 1 18.8 4.46 20 2.3 19.3 1.525.5 2.15 50 2.1 20.6 1 28.2 0.24

As the average collagen production is correlated to the number of cellsable to produce collagen, the slight difference in average collagenproduction between the first set and the second set of the study may bedue to a natural variance in the number of cells in the studied wells.

Example 4

Study of Collagen Production in Nucleus Pulposus Cells Upon Treatmentwith Lactic Acid

Culture of Human Nucleus Pulposus Cells

Nucleus pulposus (NP) cells isolated from humans (4800, ScienCell, USA)were cultured and studied. NP cells are intervertebral disc cells in thenucleus pulposus.

Firstly, cryopreserved NP cells were thawed in a 37° C. water bath. Thethawed NP cells were then suspended in supplemented Nucleus PulposusCell Medium, and thereafter seeded in a T75 cell culture flask having avolume of 75 cm³ and being coated on its inside with poly-L-lysine(0413, ScienCell, USA). The initial seeding density was 5.0×10³ viableNP cells per milliliter.

The supplemented Nucleus Pulposus Cell Medium consisted of NucleusPulposus Cell Medium (4801, ScienCell, USA) supplemented with 2% byvolume of fetal bovine serum (0010, ScienCell, USA), 1× Nucleus PulposusCell Growth Supplement (4852, ScienCell, USA) and 1×penicillin/streptomycin solution (0503, ScienCell, USA).

The T75 flask comprising the prepared NP cells was swirled to distributethe NP cells in the medium. The cell culture was thereafter incubated ina 37° C., 5% CO₂/95% air humidified cell culture incubator over night.

At confluence, the fibroblasts were diluted in the supplemented media toavoid alternations in cell phenotype, cell proliferation and/or celldifferentiation.

Preparation of Lactic Acid

Lactic acid (PURAC PF 90, Batch No. 1406001940, Corbion Purac, theNetherlands) was weighted into a sterile 10 mL tube or 50 mL tube.Milli-Q water (>18.2Ω) was added to prepare a stock solution of lacticacid. The stock solution was mixed and stored before preparing finalsolutions of lactic acid with varying concentrations. The period ofstorage was less than 1 hour at ambient temperature, or, alternatively,less than 24 hours at a temperature of 4° C.

Effect of Lactic Acid on Collagen Production in Human Nucleus PulposusCells

NP cells cultured as described above were detached from the cell cultureflask and placed on 6-well plates at an initial density of 4.5×10⁴viable cells per well. The NP cells were grown in supplemented NucleusPulposus Cell Medium. The NP cells in some of the wells were alsotreated with lactic acid (PURAC PF 90, Batch No. 1406001940, CorbionPurac, the Netherlands) in various concentrations: 0, 0.5, 5, 10, 20 and50 mg/mL, respectively (for respective pH, see table 7 above). The NPcells were incubated in a 37° C., 5% CO₂/95% air humidified cell cultureincubator for 48 hours.

To study the effect of lactic acid on the collagen production in the NPcells, a spectrophotometric method called Soluble Collagen Assay(QuickZyme Biosciences, Leiden, the Netherlands) based on binding ofSirius Red dye to collagen, was applied.

Cell media was collected from each well and 140 μL was pipetted into a96-well plate. Samples were taken in triplicates. Medium samples weremixed thoroughly with 60 μL Sirius Red dye solution by pipetting up anddown at least five times. The 96-well plate was centrifuged at 1500×gfor 2 hours. All of these steps were performed at a temperature below25° C., for example the centrifugation was performed at 4° C.

The centrifuged sample was washed and the supernatant removed. The cellpellet was resuspended in 150 μL detection solution by thoroughly mixingby pipetting up and down at least ten times. Thereafter, 100 μL of eachsample was transferred into a new 96-well plate and collagen content wasmeasured spectrophotometrically at an optical density of 540 nm.

In order to suit the apparatus of measurement, the cells were diluted inphosphate buffer solution (PBS) at a ratio of 1:1.

From the study performed in triplicate, it was clearly shown thataddition of lactic acid to NP cells increases the average production ofcollagen in the NP cells, as indicated in Table 8 and FIG. 9. Theaverage production was measured after 2 days of treatment with lacticacid.

In FIG. 9, the results from the study are presented by diamonds. Atwo-period moving average trendline has been included to schematicallyshow the trend in collagen production. The x-axis shows theconcentration of the lactic acid added to the wells comprising NP cells,and the y-axis shows the average amount of produced collagen in thesewells upon measurement after two days from addition of the lactic acidinto the wells.

More particular, the increase in production of collagen was significantwhen lactic acid was added to a concentration of at least 5 mg/mL in thewell. Further, it was shown that the collagen production increased withincreasing lactic acid concentration up to about 10-20 mg/mL, where aplateau was reached as also indicated in FIG. 9. The decrease incollagen production at 50 mg/mL is interpreted such that a treatmentwith lactic acid in such a high concentration may have cytotoxic effectscausing cell death.

TABLE 8 Effect on collagen production in NP cells of lactic acid.Concentration of Average amount of lactic acid produced collagen perStandard [mg/mL] pH well [μg] deviation 0 — 2.33 0.267 0.5 3.1 3.58 1.485 2.8 16.4 0.339 10 2.6 25.3 1.11 20 2.4 27.2 0.513 50 2.3 25.2 2.82

Example 5

Study of Collagen Production in Human Fibroblasts Upon Treatment withLactic Acid Having Differentiated pH Values

Culture of Human Fibroblasts

Adult human fibroblast cells (Detroit 551, ATCC, CCL-110) were culturedand studied.

Firstly, cryopreserved Detroit cells were thawed in a 37° C. water bath.The thawed cells were transferred to a centrifuge tube containing 9 mlEagle's Minimum essential medium 1× (Gibco Life Technologies)supplemented with non-essential amino acids (Thermo Scientific HyClone),1 mM sodium pyruvate (Thermo Scientific HyClone), 2 mM L-glutamine(Lonza) and 10% (v/v) Fetal Bovine Serum (GE Healthcare/PAA). The cellsuspension was then centrifuged at 125×g for 5 min. The cell pellet wasresuspended in 1 ml complete medium and seeded in a T75 flask containing15 ml complete medium. The cells were grown to confluence at 37° C. in ahumidified, 5% CO2 incubator. At confluence, the Detroit cells werepassaged by detaching with trypsin/versene (Thermo Scientific HycloneSV30037.01, Gibco Life Technologies 15040033). The cells were diluted incomplete medium and seeded in new culture vessels at a subcultivationratio of 1:2 to 1:5.

Preparation of Formulations

To minimize the risk of contamination, all formulations were prepared ina laminar air bench, apart from weighing of Iohexol where this was notpossible.

Formulation of IOHEXOL Solution:

(Histodenz CAS#66108-95-0 LOT #WXBB5310V) was weighted in a sterile 50ml tube. Iohexol was calculated by weight of iodine per ml of solutionexpressed as mg l/ml according to the supplied recipe. Lactic acidsolution (PURAC PF 90 Batch Nr.: 1406001940) and media was added and thesamples were mixed using an end over end rotation device for 30 minutesand were then allowed to stand refrigerated for 72 hours before settingthe pH.

Adjustment of pH:

The pH was adjusted with 1 M NaOH (Sodium hydroxide, Titrisol) or 1 MHCl (Hydrochloric acid, Titrisol).

The formulation of compounds in accordance with table 9 below.

TABLE 9 Formulation #1-8 used in the current experiment. pH NameCompound Concentration (adjusted) Media #1 Lactic acid 20 mg/ml No (pH3) Detroit 551 #2 Lactic acid 20 mg/ml to pH 3.5 Detroit 551 with NaOH#3 Lactic acid 20 mg/ml to pH 4 with Detroit 551 NaOH #4 Lactic acid 20mg/ml to pH 4.5 Detroit 551 with NaOH #5. Control 1 — — to pH 3 withDetroit 551 HCl #6. Control 2 — — to pH 3.5 Detroit 551 with HCl #7.Control 3 — — to pH 4 with Detroit 551 HCl #8. Control 4 — — to pH 4.5Detroit 551 with HClTreatment with Formulations

The Detroit cells were detached from their culture vessels and seededinto 6-well plates at a density of 80 000 cells/well. The cells weregrown in supplemented Detroit 551 medium. All formulations were dilutedin supplemented Detroit 551 medium. At day 1 the cell culture media inall wells was replaced with formulations based on lactic acid(Sigma-Aldrich, Stockholm, Sweden) described in table 1. The cells werecultured in triplicates. The cells were incubated for two days at 37° C.in a humidified, 5% CO2 incubator. The cells were treated withformulations at 80% cell confluence.

Analysis of Collagen Production

Collagen content in the cell media was measured using aspectrophotometric method analysing Sirius Red dye binding collagen(Soluble Collagen Assay, QuickZyme Biosciences, Leiden, Netherlands).Briefly, cell media was collected from each well and 140 μl was pipettedinto 96-well plates. Samples were taken in duplicates. Medium sampleswere mixed with 60 μl Sirius Red dye solution and the 96-well plate wascentrifuged at 1500×g at 4° C. for 2 hours. The pellets were resuspendedin 150 μl detection solution. Next, 100 μl of each sample wastransferred into a new 96-well plate and collagen content was measuredspectrophotometrically at an optical density of 540 nm.

pH Measurements

The pH was measured prior to treatment, 10 min after treatment and twodays after treatment with lactic acid or formulations (table 10).

TABLE 10 pH measurements in Detroit 551. pH after pH after Name StartingpH 10 min 2 days #1 7.5 3 3 #2 7.5 3.5 3.5 #3 7.5 4 4 #4 7.5 4.5 4.5 #5.Control 1 7.5 3 3 #6. Control 2 7.5 3.5 3.5 #7. Control 3 7.5 4 4 #8.Control 4 7.5 4.5 4.5

Results

No difference in cell morphology or cell density was observed at ocularinspection following treatment with formulation #1-4 as compared to thecorresponding controls #5-8. The cell viability was also inspectedocular in microscope two days after treatment and at this point of timecell media was collected and analyzed for collagen production.

The Detroit cells were seeded into 6-well plats at a density of 80 000cells/well. The cells were treated with formations at 80% cellconfluence. Collagen production was analyzed two days after addition offormulations (measurement after diluting the samples of 1:4, 1:1 andthereafter 1:1 to enable absorbance levels for the samples in the linearrange of the instrument. The data points are illustrated as averagevalues±SD in FIG. 10.

Formulations #1 and #2 gave significant collagen production. Based onthe different dilutions, formulation #1 containing 20 mg/ml lactic acidwithout any pH adjustment was the most effective with respect tocollagen production followed by #2, also containing 20 mg/ml lacticacid, but with a pH adjustment from 3.0 to 3.5, which resulted inapproximately 35% less collagen compared with formulation #1. Treatmentwith the other formulations resulted in no significant collagenproduction.

CONCLUSION

The inventors believe that the use according to embodiments of thepresent invention will treat intervertebral disc-related pain also inhumans.

The expected transformation into connective tissue of the intervertebraldisc subjected to injection of a substance, such as a lactic acid, maybe observed in vivo. Typically, the procedure will be conducted underanaesthesia or light sedation, and by using radiologic guidance. Thus,the treatment procedure will be similar to a radiologic assessment ofthe intervertebral disc, a so called discography, when a contrast mediumis injected into the intervertebral disc under radiologic guidance.

The composition should comprise lactic acid, and have a pH value below4. As seen in the results in FIG. 10, a lactic acid solution having a pHof 4 and above, will probably not result in a collagen production andaccordingly no expected transformation into connective tissue of theintervertebral disc subjected to injection of a substance comprisinglactic acid and having a pH of 4 or above.

With the present invention, the inventors, so to say, go against whathas so far been considered and presented for alleviation of disc relatedpain. While prior art is focusing on how to keep the intervertebral disc“young”, the present invention provides for an expeditious degenerationto make the disc “old”.

Various studies have shown that in older people the intervertebral discshave typically been transformed into connective tissue and willtherefore likely not give rise to pain anymore. Importantly, theliterature also demonstrates that the prevalence of discogenic pain isdrastically decreasing by the increase in age. Age-related back pain inolder people is more typically caused by osteoarthritis and/orosteoporosis, and not by the intervertebral discs. See e.g. the articlesby DePalma et al, What is the source of chronic low back pain and doesage play a role?, Pain Medicine 2011; 12:224-233, and Laplante et al,Multivariable Analysis of the Relationship Between Pain ReferralPatterns and the Source of Chronic Low Back Pain, Pain Physician 2012;15:171-178, which both disclose that discogenic low back pain is morelikely in younger patient than in older patient.

The examples in this application show that the administration of lacticacid results in a change of the tissue composition of the disc and in aflexion stiffness of the injected intervertebral discs. This might notbe a direct proof of alleviation of pain. However, it may be used as anindicator of the transformation of the nucleus pulposus into connectivetissue, which transformation will (a) stabilize the inner volume of thedisc; (b) reduce diffusion from the intervertebral disc; and (c) reducemicro motions in the disc, which all will contribute to reduceintervertebral disc-related pain.

Increased levels of lactic acid in the intervertebral disc are known tocause degeneration of the disc, and such degeneration causes back pain.In the natural degeneration of the disc, the nucleus pulposus willslowly transform to connective tissue from a state where it initiallycauses pain, to a state where the pain is relieved, see for exampleKirkaldy-Willis et al, Instability of the Lumbar Spine, ClinicalOrthopaedics and Related Research, No. 165, May 1982, pages 110-123, andespecially page 123, upper right column. Such transformation will takeabout 20 to 40 years without any treatment. By injecting considerablyhigher concentrations of lactic acid than those found in an initialstage of degeneration, the time it takes for the transformation of thenucleus pulposus into connective tissue will decrease from the 20-40years (without treatment) to about 4 weeks.

Other substances capable of inducing accelerated degeneration of anintervertebral disc may also be considered as substitutes and/oralternatives to lactic acid.

1. A method for treating intervertebral disc-related pain, the methodcomprising: administering a therapeutically-effective amount of acomposition comprising lactic acid and having a pH below 4 into a discspace comprising the nucleus pulposus of an intervertebral disc in apatient in need thereof.
 2. The method according to claim 1, wherein thecomposition is administrated in an amount effective to increase theconcentration of lactic acid in said disc space to above 12 mmol/L. 3.The method according to claim 1, wherein the concentration of lacticacid in the composition is at least 12 mmol/L.
 4. The method accordingto claim 1, wherein said composition has a pH below 3.5.
 5. The methodaccording to claim 1, wherein said composition has a pH below 3.0. 6.The method according to claim 1, wherein said composition isadministered to the disc space of an intervertebral disc contributing tothe intervertebral disc-related pain.
 7. The method according to claim1, wherein said composition is administered by local injection into thedisc space comprising the nucleus pulposus.
 8. The method, according toclaim 1, wherein the lactic acid is administered in a single dosagewithin the range of from 2 mg to 200 mg.
 9. The method according toclaim 8, Wherein said lactic acid is administered at a single occasionin said single dosage.
 10. The method according to claim 1, wherein saidcomposition is in the form of an aqueous solution comprising said lacticacid.
 11. The method according to claim 1, wherein said intervertebraldisc-related pain is at least one selected from the group consisting ofneck pain, chronic neck pain, low back pain, and chronic low back pain.12. The method according to claim 1, wherein said intervertebraldisc-related pain is coccygodynia.
 13. The method according to claim 1,wherein a flexion stiffness of the intervertebral disc is higher afteradministering the composition.
 14. The method according to claim 1,wherein the nucleus pulposus of the intervertebral disc is transformedinto a solid connective tissue.
 15. The method of claim 14, wherein thesolid connective tissue comprises collagen.
 16. A method for increasinga flexion stiffness of an intervertebral disc, the method comprisingadministering a composition comprising lactic acid and having a pH below4 into a disc space comprising the nucleus pulposus of theintervertebral disc in a patient in need thereof.
 17. A method fortransforming a nucleus pulposus of an intervertebral disc into a solidconnective tissue, the method comprising administering a compositioncomprising lactic acid and having a pH below 4 into a disc spacecomprising the nucleus pulposus of the intervertebral disc in a patientin need thereof.
 18. The method of claim 16, wherein the solidconnective tissue comprises collagen.
 19. The method according to claim1, wherein the lactic acid is administered in a single dosage within therange of from 10 mg to 100 mg.
 20. The method according to claim 1,wherein the lactic acid is administered in a single dosage within therange of from 10 mg to 50 mg.